Translation Flashcards
Amino acid binding to tRNA
- Binding of an amino acid to a particular tRNA establishes the genetic code
- Binding of amino acid to tRNA activates the amino acid
- Activation needed because formation of peptide bond between free amino acids is unfavourable
- Activation forms amino acid ester
- Activation done by specific aminoacyl-tRNA synthetase enzymes using 2 ATP
Amino acid + ATP + tRNA + H2O –>
aminoacyl-tRNA + AMP + 2 Pi
The enzyme responsible for this activation process is aminoacyl-tRNA synthetase. It catalyzes the reaction using two ATP molecules, which provide the energy required for bond formation.
The aminoacyl-tRNA synthetase enzyme facilitates the binding of a specific amino acid to its corresponding tRNA, using the energy from the hydrolysis of ATP. The products are an aminoacyl-tRNA (the activated amino acid attached to its tRNA), adenosine monophosphate (AMP), and two inorganic phosphate ions (Pi).
**unfavourable reaction **: energy is not lost during ATP hydrolysis and it’s stored in the form of a high-energy bond that links the amino acid to the tRNA.
Later, when protein synthesis takes place in the ribosome, this high-energy bond is broken, and the stored energy is released. This released energy then serves as the activation energy for the formation of a peptide bond, linking the amino acid to the growing protein chain.
Amino acid recognition by aminoacyl-tRNA synthetase
Aminoacyl-tRNA synthetase is an enzyme
The process involves two steps:
- The enzyme binds to the amino acid and ATP, catalyzing a reaction that results in the formation of an aminoacyl-adenylate intermediate and the release of inorganic phosphate. This step activates the amino acid for the following step.
- The enzyme then catalyzes the transfer of the activated amino acid to the appropriate tRNA molecule.
proofreading :
However, mistakes can occur, and an incorrect amino acid might be attached to the tRNA. This is where the editing site comes in.
The editing site is a secondary active site in the aminoacyl-tRNA synthetase that can recognize and remove incorrectly attached amino acids. So, if the wrong amino acid is attached to the tRNA, the molecule will fit into the editing site, and the incorrect amino acid will be cleaved off, ensuring that only the correct amino acid remains attached to the tRNA.
e.g. when serine is incorrectly attached to threonyl-tRNA, it will be cleaved off so threonine can attach instead to tRNA.
ribosomes
Ribosomes bind tRNA and mRNA
Ribosomes have a large subunit (50s) and small subunit (30s)
- Ribosomes have 3 tRNA-binding sites that span the
50S and 30S subunits: the Aminoacyl, Peptidyl and
Exit sites - mRNA is bound within the 30S subunit
- tRNA in the A and P sites are bound to mRNA via
anticodon-codon pairing
tranlsation in prokaryotes
prokaryotic transcription and translation are closely coupled in space and time
==> happen in the same location within the cell, almost simultaneously.
- The 5’ end of mRNA interacts with ribosomes way before transcription of the 3’ end is finished
–> As soon as the 5’ end of the mRNA molecule (which is the start of the transcribed message) is synthesized, it can begin to bind with ribosomes. This is while the rest of the mRNA molecule is still being transcribed from the DNA template. - Possible because both transcription and translation proceed in the 5’ to 3’ direction
Multiple ribosomes translating an mRNA strand
== One single mRNA molecule can be read by multiple ribosomes at the same time, each synthesizing a separate protein molecule.
initaition of translatio
Translation, the process of protein synthesis from an mRNA template, doesn’t start right at the beginning of the mRNA. Instead, it starts at a specific sequence known as the start codon.
Translation is initiated at the start codon, which is usually found more than 25 nucleotides away from the 5’ end of the mRNA. This region between the 5’ end and the start codon is known as the 5’ untranslated region (5’ UTR).
In bacterial cells, many mRNA molecules are ‘polycistronic’, meaning they contain the information for more than one protein. This contrasts with eukaryotic cells where most mRNA molecules are ‘monocistronic’ and code for a single protein.
Each gene within a polycistronic mRNA has its own start and stop codons which serve as signals for the ribosome to start and stop translation. This means that each protein within a polycistronic mRNA is synthesized independently of the others.
The lac operon is an example of a group of genes in bacteria that are transcribed together into a single polycistronic mRNA molecule. The proteins encoded by these genes are involved in lactose metabolism.
Initiation of translation – start signals
The initiator codon marks the start of the region on the mRNA that will be translated into protein. In prokaryotes, the initiator codon is typically AUG, which codes for methionine.
The initiator codon pairs with its matching anticodon, which is a sequence of three nucleotides on tRNA. The first tRNA carries the amino acid Methionine, and its anticodon is complementary to the AUG initiator codon.
The Shine-Dalgarno sequence is a purine-rich region located approximately 10 nucleotides upstream of the initiator codon. This sequence helps to recruit the ribosome to the correct starting point on the mRNA.
The Shine-Dalgarno sequence pairs with a complementary region in the 16S rRNA of the small ribosomal subunit. This interaction helps to position the ribosome correctly at the initiation codon to start protein synthesis.
The regions of the mRNA molecule are divided into ‘translated’ and ‘untranslated’ regions. The translated region contains the actual genetic code for the protein, while the untranslated regions (at both ends of the mRNA) contain regulatory sequences that control translation.
Initiation of translation - fMet
In bacteria, the first amino acid incorporated into a growing polypeptide chain during protein synthesis is not methionine, but a modified form known as N-formylmethionine (fMet).
The initiation of protein synthesis relies on a special initiator tRNA that recognises the AUG start codon on the mRNA. This tRNA carries the modified amino acid fMet.
The tRNA that delivers fMet at the start of translation is different from the tRNA that adds methionine in the middle of the polypeptide chain during elongation.
This indicates that only the methionine carried by the initiator tRNA gets modified to fMet. Methionine incorporated later during the protein synthesis is not modified.
After the new polypeptide chain has started to form and reached about 10 amino acids in length, the fMet at the start of the chain is removed in approximately half of all proteins.
Both the initiator tRNA, which brings the first amino acid (fMet) to the mRNA, and the regular Met tRNA, which adds methionine in the middle of the polypeptide chain, are charged by the same enzyme - an aminoacyl-tRNA synthetase.
The methionine attached to the initiator tRNA is modified by an enzyme called transformylase. The process of adding a formyl group to methionine is known as formylation, hence the product is formylmethionine (fMet).
This point emphasizes that transformylase is specific - it only modifies the methionine that is attached to the initiator tRNA. It does not modify free methionine or methionine carried by the regular Met tRNA.
Shine-Dalgarno sequence
The binding of the Shine-Dalgarno sequence to the 16S rRNA in the ribosomal small subunit is a crucial step in the initiation of translation in bacteria. The Shine-Dalgarno sequence is a specific sequence of nucleotides found on the mRNA, and the 16S rRNA is part of the small subunit of the ribosome.
This binding interaction ensures that the ribosome is correctly positioned to start translating the mRNA from the appropriate place - the start codon. Without this interaction, the ribosome could start translating at any point along the mRNA, resulting in a non-functional protein.
In other words, the Shine-Dalgarno sequence is like a ‘landing pad’ for the ribosome on the mRNA, ensuring that translation starts from the correct position.
Initiation factors
Initiation factors are proteins that help start the process of translation. They bind to the small 30S ribosomal subunit and prevent it from associating with the large 50S subunit prematurely.
The initiation factors, along with the initiator tRNA and mRNA, come together with the 30S ribosomal subunit to form a complex known as the 30S initiation complex. Here, the mRNA binds to the rRNA in the 30S subunit, and the initiator tRNA binds to the AUG start codon on the mRNA and to the P site on the 30S subunit.
Once the 30S initiation complex is assembled, the large 50S subunit can bind, forming the complete 70S ribosome. This step is the rate-limiting step, meaning it’s the slowest and most regulated step in the process.
Q: What is the starting point for the elongation phase in translation?
A: The starting point for the elongation phase in translation is when the P site of the ribosome has the initiator tRNA and the A and E sites are empty.
Q: Which elongation factor is responsible for the delivery of the next tRNA to the A site?
A: The elongation factor Tu (EF-Tu) is responsible for the delivery of the next tRNA to the A site.
Q: What is the role of EF-Tu in the translation process?
A: EF-Tu has multiple roles: it protects the ester bond in the activated amino acid, it checks the accuracy of the anticodon-codon binding, and it binds to all tRNA except the initiator tRNA.
Q: Where is the peptide bond formation catalyzed during elongation?
A: The formation of the peptide bond is catalyzed by rRNA in the large 50S ribosomal subunit.
Q: What process moves the tRNA and mRNA by 3 nucleotides in the small subunit during elongation?
A: The elongation factor G (EF-G) helps in a process called translocation, which moves the tRNA and mRNA by 3 nucleotides in the small subunit.
Q: In what direction are proteins made?
A: Proteins are made in the N to C terminal direction.
